WO2023181067A1 - A process for improving biological nitrogen fixation by microbes beneficial to crops - Google Patents
A process for improving biological nitrogen fixation by microbes beneficial to crops Download PDFInfo
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- WO2023181067A1 WO2023181067A1 PCT/IN2023/050271 IN2023050271W WO2023181067A1 WO 2023181067 A1 WO2023181067 A1 WO 2023181067A1 IN 2023050271 W IN2023050271 W IN 2023050271W WO 2023181067 A1 WO2023181067 A1 WO 2023181067A1
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- ammonia
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
- C12Y603/01—Acid-ammonia (or amine)ligases (amide synthases)(6.3.1)
- C12Y603/01002—Glutamate-ammonia ligase (6.3.1.2)
Definitions
- the present invention relates to a process for genetic modification of microbes for improved Nitrogen fixation.
- the present invention relates to a process for gene modification of microbes to reduce glucose uptake by the microbes and in turn increase the ammonia availability to the plants.
- the present invention also relates to Down regulation or deletion of glnA and amtB in Nitrogen fixing microbes along with/without inhibition of glucose uptake increased the Nitrogen fixation efficiency of microbes.
- the present invention relates to over expression of nifA genes (to enhance the Nitrogenase activity) and glutaminase (hydrolyses glutamine to glutamate and ammonia) for increased Nitrogen fixation.
- the present invention relates to over expression of the native nitrogenase (to enhance the Nitrogenase activity) for increased Nitrogen fixation.
- the present unvention also relates to hetelogus expression of Fe-Nirogenase from the Diazotrophic microorganisms such as Azotobacter,Rhodobacter etc and not limited to these microorganisms
- the present invention also relates to make the Diazotroph become autotrophic by introduction of pathways related to utilization of Cl compounds such as Methane, CO2, Formaldehyde, Methanol etc as souce of carbon and energy
- the present invention relates to a process of CRISPR/Cas technology used preferentially for gene manipulation of microbes.
- Plants synthesize glucose in sunlight with the help of chlorophyll, carbon dioxide and water and given away oxygen. It can be changed over into synthetic compounds (cellulose) required for development of plant cells or it can be changed over into starch, a capacity particle that can be changed over back to glucose when the plant requires it.
- synthetic compounds cellulose
- Nitrogen is an important component of all proteins, enzymes and nucleic acids of plants and animals. It is a component of chlorophyll in plants. Even though it is abundantly found in nature, much of this nitrogen is inert and cannot be used by the plants. In order for the plants to use this nitrogen it has to be converted into Nitrate and Ammonia. This can be achieved either manually by adding ammonia or nitrate fertilizer to the soil or through biological nitrogen fixation.
- the biological nitrogen fixation is done by microorganisms which express an enzyme called nitrogenase that combines the gaseous, atmospheric nitrogen with hydrogen to produce ammonia.
- the process is carried out by two main types of microorganism: those which live in close symbiotic association with other plants and those which are “free living” or non-symbiotic.
- the plant provides glucose to the nitrogen-fixing microorganism that are utilized by them for the energy they need for nitrogen fixation.
- the microbes provides fixed nitrogen to the host plant for its growth.
- microbes use host plants for their growth, colonization, and proliferation; however, they offer a variety of benefits to the hosts.
- Colonization of microorganisms on host plants takes place through air, water, and insects, or they may also be present in germinating plant parts. Endophytic microbial interactions influence the internal part, while epiphytic microbial interactions influence the exterior surface of the plants.
- Rhizospheric freeliving microbes grows with the help of plant exudates. These microbes are not harmful to the plants; however, they secrete some beneficial substances which may help in plant growth promotion, resistance to pathogenic microbes, removal of harmful contaminants, and production of secondary metabolites. In such a way, microbes contribute in agricultural crop improvement.
- US20200331820A1 discloses a genetically engineered bacterium comprising a modification in glnD gene and a genetically engineered bacterium comprising a modification in glnA gene by replacing the native promoter of glnA with a promoter with lower activity which results in decreased expression or activity of glnA.
- Nitrogen fixing endophytes, ephiphytes and root endophytes depend up on the glucose present in the plants. But glucose inhibits the ammonia accumulation. Therefore, there is a need in the art to provide for genetic modifications of microbes for improved Nitrogen fixation by reducing Glucose uptake and increasing ammonia accumulation in plants.
- composition of organisms includes strains such as Methylobacterium, Azotobacter modified for efficient Nitrogen fixation and application in foliar or soil applications
- the present invention provides a process for gene modification of microbes to reduce glucose uptake and increase ammonia availability to the plants by the microbes.
- the present invention provides a process for genetic modification of micro-organisms, wherein a reduced glucose uptake and improved Nitrogen fixation is facilitated.
- the invention provides a process of CRISPR/Cas technology used prefentially for gene manipulation of microbes.
- the Glucose uptake is reduced by disruption of ptsG gene (glucose transporter) and the amino acid catabolism for improved nitrogen fixation is promoted by disruption of glnA gene (glutamine synthetase) and amtB gene (ammonia transporter to inside the bacterial cell).
- the present invention provides gene modification in microbes that result in increased expression of nifA genes (to enhance the Nitrogenase activity) and glutaminase (hydrolyses glutamine to glutamate and ammonia) for increased Nitrogen fixation.
- the present invention provides genetically modified microbes favourable for reducing glucose uptake by microbes and increasing ammonia assimilation by plants.
- the present invention provides the involvement of CRISPR/Cas technology used preferentially for gene manipulation of microbes.
- Figure 1 Depicts impact of downregulation of Ammonia uptake process on increased released of Ammonia.
- Figure 2 Depicts impact of enhanced activity of Nitrogenase and related factors, on increased Nitrogen fixation and Ammonia release.
- Figure 3 Depicts impact of different carbon metabolism of Cl compounds on increased carbon metabolism.
- the present invention provides a process for the gene modification of microbes to reduce glucose uptake by the microbes and increase ammonia accumulation by the plants.
- the present inventon provides a process for the gene modification of microbes to reduce glucose uptake by microbes and increase ammonia accumulation by plants wherein glucose uptake reduction is facilitated by ptsG gene (glucose transporter) disruption and amino acid catabolism is promoted by glnA gene (glutamine synthetase) disruption and amtB gene (ammonia transporter) disruption.
- ptsG gene glucose transporter
- amino acid catabolism is promoted by glnA gene (glutamine synthetase) disruption and amtB gene (ammonia transporter) disruption.
- the present invention provides gene manipulations in microbes selected from one or more of endophytic/ epiphytic/ rhizospheric microbes that results in reduced Glucose uptake by microbes and increased ammonia accumulation by plants.
- the present invention provides down regulation or deletion of gln and amtB gene in Nitrogen fixing microbes along with/without inhibition of glucose uptake resulting in increased Nitrogen fixation efficiency of the microbes.
- Glucose transporter gene /v.s gene. glutamine synthase gene, glnA gene and Ammonia transporter gene, amtB gene were disrupted resulting in reduced glucose uptake by microbes and enhanced amino acid catabolism.
- the gene modification results in decreased expression of glnA.
- the gene modification comprises replacing the native promoter of glnA with a promoter with lower activity.
- the genetically engineered bacterium comprising a modification in glnA which results in decreased expression or activity of glnA.
- the present invention provides gene modification in microbes that result in over expression of nifA genes (to enhance the Nitrogenase activity) and glutaminase (hydrolyses glutamine to glutamate and ammonia) for increased Nitrogen fixation.
- the present invention provides a composition comprising genetically modified micro-organism(s) consisting of upregulation of nifA and iron containing Nitrogenase and downregulation of glutaminase gene for increased Nitrogen fixation wherein the said micro-organism is an endophyte, an epiphyte and a rhizospheric microbe.
- the present invention provides a composition comprising genetically modified micro-organism(s) consisting of down regulation or deletion of glnA and amtB gene.
- the present invention provides a composition comprising genetically modified micro-organism(s) consisting of disruption of /V.sG gene, glutamine synthase gene, glnA gene and Ammonia transporter gene, amtB gene.
- the present invention provides a plasmid vector or suicidal plasmid consisting of integration site as well as over expression genes along with strong constitutive promoter.
- the plasmid vector is carrying a DNA construct comprising a nucleotide sequence represented by SEQ ID NO.9.
- the present invention provides SEQ ID NO. 9. consisting of 5119 bp with Fe-Nitrogenase with strong promoter nifH (SEQ ID NO: 8) and integration site sequence formate dismutase with SEQ ID NO: 6 and 7.
- the plasmid vector is carrying a DNA construct comprising a nucleotide sequence represented by SEQ ID NO: 1.
- the plasmid vector is expressed in miro-organism selected from the group comprising of endophytic bacteria, epiphytic bacteria, rhizospheric bacteria and fungi.
- the bacteria is selected from the group comprising ofcultivable bacteris (viz) Methylobacterium extorquens; Beijerinckia indica; Azoarchus communis and uncultivable bacteria adapted to cultivabe using Ichip method (viz) Pseudomonas sp.; Bacillus sp. and Sphingomonas sp.
- cultivable bacteris viz) Methylobacterium extorquens; Beijerinckia indica
- Azoarchus communis and uncultivable bacteria adapted to cultivabe using Ichip method viz) Pseudomonas sp.; Bacillus sp. and Sphingomonas sp.
- the present invention provides a process for the preparation of genetically modified micro-organism(s) for improved nitrogen fixation and its delivery to crop-plants for assimilation, wherein the said microorganism is an endophyte, an epiphyte and a rhizospheric microbe, wherein the said micro-organism is uncharacterized and non-cultivated, wherein the said modiciation is carried out by homologous recombination
- the present invention provides microbes selected from nitrogen fixing bacteria.
- the Nitrogen fixing bacteria may include Proteobacteria (such as Methylobacter, Erwinia, Acinetobacter, Beijernickia, Sphingomona, Novosphingobium, Ochrobactrum, Gluconacetobacter etc.), Firmicutes (such as Clostridium sp. etc.), Cyanobacteria (such as cyanobacteria sp.), Actinobacteria (such as Frankia, Arthrobacter, Agromyces, Corynebacterium, Mycobacterium, Micromonospora, Propionibacteria etc.) and Bacteroidetes (such as Flavobacterium etc).
- Proteobacteria such as Methylobacter, Erwinia, Acinetobacter, Beijernickia, Sphingomona, Novosphingobium, Ochrobactrum, Gluconacetobacter etc.
- Firmicutes such as Clostridium sp. etc.
- the present invention provides the genetic modification of micro-organisms for improving Nitrogen fixation in plants.
- the present invention provides genetically modifies microbes, wherein glucose uptake reduction is facilitated by ptsG gene (glucose transporter) disruption and amino acid catabolism is promoted by glnA gene (glutamine synthetase) disruption.
- the microorganisms can be selected from but not limited to Methylobacterium, Methyloversatilis, Sphingomonas, Bosea, Altererythrobacter, Brevundimonas, Rubrivivax, Niveispirillum, Dinoroseobacter shibae.
- Other microbes includes Nitrosopumilus maritimus , Nitrospira inopinata, Rhodobacter sphaeroides, Cupriavidus necator, Nigrospora oryzae, Azospirillum lipoferum, Rhodopseudomonas palustris , Bradyrhizobium japonicum , Ralstonia eutropha , Cyanobacteria, Epichloe typhina , Rhodococcus ,Xanthobacter species Nitrosomonas, Nitrosospira, Nitrosococcus, and Nitrosolobus .
- the present invention provides certain fungi comprising Aspergillus, Candida, Chlamydomonas, Chrysosporium, Cryotococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora, Penicillium (e.g. P. chrysogenum), Pichia, Saccharomyces, Trichoderma and Xanthophyllomyces to be selected for genetic modification.
- fungi comprising Aspergillus, Candida, Chlamydomonas, Chrysosporium, Cryotococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora, Penicillium (e.g. P. chrysogenum), Pichia, Saccharomyces, Trichoderma and Xanthophyllomyces to be selected for genetic modification.
- Coli Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, Zymomonas and Saccharomyces, e.g., Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Kluyveromyces lactis, Saccharomyces lactiss.
- the present invention provides endophytes selected from the group comprising Proteobacteria (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Se"atia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Delftia, Bradyrhizobiun, Sinorhizobium and Halomonas)' , Firmicutes (such as Bacillus, Paenibacillus, Lactobacil/us, Mycoplasma, and Acetabacterium) and Actinobacteria (such as Streptomyces, Rhodacoccus, Microbacterium, and Curtobacterium).
- Proteobacteria such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Panto
- Bacteria that can be produced by the methods disclosed herein include Azotobacter sp., Bradyrhizobium sp., Klebsiella sp., and Sinorhizobium sp.
- the bacteria may be selected from the group consisting of Azotobacter vinelandii or Azotobacter chroococcum, Bradyrhizobium japonicum, Klebsiella pneumoniae, and Sinorhizobium meliloti.
- the bacteria may be of the genus Enterobacter and Rahnella.
- certain fungi comprising Saccharomyces cerevisiae and Trichoderma harzianum are selected for genetic modification for nitrogen fixation.
- the present invention provides a process for gene modification of microbes that reduces glucose transport and increases ammonia accumulation in crops by CRISPR/Cas technology.
- the present invention provides release of ammonia to the environment by the microbes in absence of glucose uptake.
- Example 1 Down regulation/ Deletion of glnA, ptsG and amtB genes
- Nitrogen fixation in diazotrophs was improved by reducing glucose uptake and increasing ammonia accumulation.
- the gene modification comprised replacing the native promoter with a promoter with lower activity. This resulted in decreased expression or activity of these enzymes and thereby reduced glucose uptake and improved Nitrogen fixation and nitrate uptake by the plant.
- Example 2 Impact of downregulation of Ammonia uptake process on increased released of Ammonia: Inorder to increase the amount of Ammonia release from the cell, internal pathways which involve Ammonia uptake were manipulated. This includes Ammonia transporter for uptake of external ammonia and the Glutaminase/Glutamine synthase involved in fixing Ammonia in Amino acid Glutamine. Downregulation of genes was achieved by promoter exchange strategy, which resulted in 2 fold increase in Ammonia release by final strain when compared to wild type strain (figure 1).
- Example 3 Enhancement of the Nitrogenase activity and regulator, for increased Nitrogen fixation in diazotrophs for enhanced Ammonia release to plants.
- Fe-Nitrogenase (Sequece ID: 1) from microbes such as Azotobacter, Rhodobacter sp., Methylobacterium spp., for facilitating Molybdenum independent Nitrogenase activity.
- protein with highest nitrogenous activity was mined from available database using NCBI data server and bioinformatic approaches such as BLAST.
- Expression cassestte was integrated into Methylobacterium spp., genome using homologous recombination at formaldehyde dismutase (fdm) inorder to delete the fdm sequence as well as simultaneous integration of Fe -nitrogenasegene cassette.
- fdm formaldehyde dismutase
- Expression cassette was cloned inside Nael restriction digestion sequences available in fdm between to get the final integration cassette (Sequence ID: 9).
- Methylobacterium spp. electrocompetent cells were transformed with Integration cassette plasmid by standard methods
- Engineered strains were subjected to growth and Ammpnia Production assay at controlled conditions. Wild type cells were also experimented in similar conditions, to test the increased Ammonia production by using Nessler reagent.
- Manipulations also include overexpression/ multi copy expression of Nitrogenase controller protein such as NifA. All these manipulations were incorporated on strain with down regulated Gin activity, and finally resulted in almost 4 fold increase in N 2 fixation, in terms of Ammonia production.
- Nitrogenase controller protein such as NifA. All these manipulations were incorporated on strain with down regulated Gin activity, and finally resulted in almost 4 fold increase in N 2 fixation, in terms of Ammonia production.
- N2 fixation by Nitrogenase depends on additional regulation controls such as NifA regulator. Increasing the Nitrogenase activity, in particular, Fe-Nitrogenase activity and multicopy expression of NifA resulted in more than 4 folds increase in N2 fixation in terms of Ammonia release (in mM) (figure 2 ).
- Example 5 Enablement of Autorophism by introduction of pathways related to utilization of Cl compound.
- Gene modification include incorporation/ overexpression of enzymes involved in methane assimilation, such as Methane monooxygenase, alcohol oxidase/ dehydrogenase and CO2 fixation via Formate dehydrogenase (FDH), Rubisco, and entry into regular metabolic pathways, finally resulting in ATP, NADH generation.
- enzymes involved in methane assimilation such as Methane monooxygenase, alcohol oxidase/ dehydrogenase and CO2 fixation via Formate dehydrogenase (FDH), Rubisco, and entry into regular metabolic pathways, finally resulting in ATP, NADH generation.
- FDH Formate dehydrogenase
Abstract
The present invention provides genetic modification of microbes for improved Nitrogen fixation. More particularly, the present invention provides down regulation or deletion of glnA and amtB genes and over expression of nifA genes and glutaminase to increase the Nitrogen fixation efficiency of microbes.
Description
“A process for improving biological Nitrogen fixation by microbes beneficial to crops”
TECHNICAL FIELD OF THE INVENTION:
The present invention relates to a process for genetic modification of microbes for improved Nitrogen fixation.
The present invention relates to a process for gene modification of microbes to reduce glucose uptake by the microbes and in turn increase the ammonia availability to the plants.
Further, the present invention also relates to Down regulation or deletion of glnA and amtB in Nitrogen fixing microbes along with/without inhibition of glucose uptake increased the Nitrogen fixation efficiency of microbes.
Further, the present invention relates to over expression of nifA genes (to enhance the Nitrogenase activity) and glutaminase (hydrolyses glutamine to glutamate and ammonia) for increased Nitrogen fixation.
Further, the present invention relates to over expression of the native nitrogenase (to enhance the Nitrogenase activity) for increased Nitrogen fixation.
Further, the present unvention also relates to hetelogus expression of Fe-Nirogenase from the Diazotrophic microorganisms such as Azotobacter,Rhodobacter etc and not limited to these microorganisms
The present invention also relates to make the Diazotroph become autotrophic by introduction of pathways related to utilization of Cl compounds such as Methane, CO2, Formaldehyde, Methanol etc as souce of carbon and energy
Further, the present invention relates to a process of CRISPR/Cas technology used preferentially for gene manipulation of microbes.
BACKGROUND AND PRIOR ART OF THE INVENTION:
Plants synthesize glucose in sunlight with the help of chlorophyll, carbon dioxide and water and given away oxygen. It can be changed over into synthetic compounds (cellulose) required for development of plant cells or it can be changed over into starch, a capacity particle that can be changed over back to glucose when the plant requires it.
Nitrogen is an important component of all proteins, enzymes and nucleic acids of plants and animals. It is a component of chlorophyll in plants. Even though it is abundantly found in nature, much of this nitrogen is inert and cannot be used by the plants. In order for the plants to use this nitrogen it has to be converted into Nitrate and Ammonia. This can be achieved either manually by adding ammonia or nitrate fertilizer to the soil or through biological nitrogen fixation.
The biological nitrogen fixation is done by microorganisms which express an enzyme called nitrogenase that combines the gaseous, atmospheric nitrogen with hydrogen to produce ammonia. The process is carried out by two main types of microorganism: those which live in close symbiotic association with other plants and those which are “free living” or non-symbiotic.
The plant provides glucose to the nitrogen-fixing microorganism that are utilized by them for the energy they need for nitrogen fixation. In exchange for these carbon sources, the microbes provides fixed nitrogen to the host plant for its growth.
These microbes use host plants for their growth, colonization, and proliferation; however, they offer a variety of benefits to the hosts. Colonization of microorganisms on host plants takes place through air, water, and insects, or they
may also be present in germinating plant parts. Endophytic microbial interactions influence the internal part, while epiphytic microbial interactions influence the exterior surface of the plants. Rhizospheric freeliving microbes grows with the help of plant exudates. These microbes are not harmful to the plants; however, they secrete some beneficial substances which may help in plant growth promotion, resistance to pathogenic microbes, removal of harmful contaminants, and production of secondary metabolites. In such a way, microbes contribute in agricultural crop improvement.
US20200331820A1 discloses a genetically engineered bacterium comprising a modification in glnD gene and a genetically engineered bacterium comprising a modification in glnA gene by replacing the native promoter of glnA with a promoter with lower activity which results in decreased expression or activity of glnA. Nitrogen fixing endophytes, ephiphytes and root endophytes depend up on the glucose present in the plants. But glucose inhibits the ammonia accumulation. Therefore, there is a need in the art to provide for genetic modifications of microbes for improved Nitrogen fixation by reducing Glucose uptake and increasing ammonia accumulation in plants.
OBJECT OF THE INVENTION:
It is an object of the present invention to provide a process for gene modification of microbes to reduce glucose uptake by the microbes and increase ammonia availability to the plants by the microbes.
It is a further object of the present invention to provide a process of gene modification of microbes that reduces glucose transport and increases ammonia accumulation in crops by CRISPR/Cas technology.
It is another object of the present invention to provide for release of ammonia to the environment by the microbes in absence of glucose uptake.
It is another object of the present invention to provide the Down regulation or deletion of glnA and amtB gene in Nitrogen fixing microbes along with/without inhibition of glucose uptake resulting in increased Nitrogen fixation efficiency of microbes.
It is a further object of the invention to provide for over expression of nifA genes (to enhance the Nitrogenase activity) and glutaminase (hydrolyses glutamine to glutamate and ammonia) for increased Nitrogen fixation.
It is another object of the present invention to provide a process for gene modification of microbes from one or more of endophytic, epiphytic or rhizosperic micro-organisms to reduce glucose uptake by the microbes and increase ammonia accumulation by the plants.
It is an object of the present invention to provide genetically modified microbes favourable for fixing atmospheric nitrogen and increasing ammonia accumulation by plants.
It is yet another object of the present invention to provide a composition comprising genetically modified microbes which are favourable for fixing atmospheric nitrogen and increasing ammonia accumulation by plants. In particular, the composition of organisms includes strains such as Methylobacterium, Azotobacter modified for efficient Nitrogen fixation and application in foliar or soil applications
It is a further object of the present invention to provide a process of CRISPR/Cas technology used preferentially for gene manipulation of microbes.
SUMMARY OF THE INVENTION:
In an aspect, the present invention provides a process for gene modification of microbes to reduce glucose uptake and increase ammonia availability to the plants by the microbes.
In another aspect, the present invention provides a process for genetic modification of micro-organisms, wherein a reduced glucose uptake and improved Nitrogen fixation is facilitated.
Accordingly, the invention provides a process of CRISPR/Cas technology used prefentially for gene manipulation of microbes.
Accordingly, the Glucose uptake is reduced by disruption of ptsG gene (glucose transporter) and the amino acid catabolism for improved nitrogen fixation is promoted by disruption of glnA gene (glutamine synthetase) and amtB gene (ammonia transporter to inside the bacterial cell).
In yet another aspect, the present invention provides gene modification in microbes that result in increased expression of nifA genes (to enhance the Nitrogenase activity) and glutaminase (hydrolyses glutamine to glutamate and ammonia) for increased Nitrogen fixation.
In another aspect, the present invention provides genetically modified microbes favourable for reducing glucose uptake by microbes and increasing ammonia assimilation by plants.
In yet another aspect, the present invention provides the involvement of CRISPR/Cas technology used preferentially for gene manipulation of microbes.
BRIEF DESCRIPTION OF THE DRAWINGS:
Figure 1: Depicts impact of downregulation of Ammonia uptake process on increased released of Ammonia.
Figure 2: Depicts impact of enhanced activity of Nitrogenase and related factors, on increased Nitrogen fixation and Ammonia release.
Figure 3: Depicts impact of different carbon metabolism of Cl compounds on increased carbon metabolism.
DETAILED DESCRIPTION OF THE INVENTION
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.
In a preferred embodiment, the present invention provides a process for the gene modification of microbes to reduce glucose uptake by the microbes and increase ammonia accumulation by the plants.
In one embodiment, the present inventon provides a process for the gene modification of microbes to reduce glucose uptake by microbes and increase ammonia accumulation by plants wherein glucose uptake reduction is facilitated by ptsG gene (glucose transporter) disruption and amino acid catabolism is promoted by glnA gene (glutamine synthetase) disruption and amtB gene (ammonia transporter) disruption.
In a preferred embodiment, the present invention provides gene manipulations in microbes selected from one or more of endophytic/ epiphytic/ rhizospheric microbes that results in reduced Glucose uptake by microbes and increased ammonia accumulation by plants.
In another embodiment, the present invention provides down regulation or deletion of gln and amtB gene in Nitrogen fixing microbes along with/without
inhibition of glucose uptake resulting in increased Nitrogen fixation efficiency of the microbes.
Accordingly, Glucose transporter gene, /v.s gene. glutamine synthase gene, glnA gene and Ammonia transporter gene, amtB gene were disrupted resulting in reduced glucose uptake by microbes and enhanced amino acid catabolism.
In some embodiments, the gene modification results in decreased expression of glnA.
In some embodiments, the gene modification comprises replacing the native promoter of glnA with a promoter with lower activity.
In some embodiments, the genetically engineered bacterium comprising a modification in glnA which results in decreased expression or activity of glnA.
In another embodiment, the present invention provides gene modification in microbes that result in over expression of nifA genes (to enhance the Nitrogenase activity) and glutaminase (hydrolyses glutamine to glutamate and ammonia) for increased Nitrogen fixation.
In another embodiment, the present invention provides a composition comprising genetically modified micro-organism(s) consisting of upregulation of nifA and iron containing Nitrogenase and downregulation of glutaminase gene for increased Nitrogen fixation wherein the said micro-organism is an endophyte, an epiphyte and a rhizospheric microbe.
In another embodiment, the present invention provides a composition comprising genetically modified micro-organism(s) consisting of down regulation or deletion of glnA and amtB gene.
In another embodiment, the present invention provides a composition comprising genetically modified micro-organism(s) consisting of disruption of /V.sG gene, glutamine synthase gene, glnA gene and Ammonia transporter gene, amtB gene.
In another embodiment, the present invention provides a plasmid vector or suicidal plasmid consisting of integration site as well as over expression genes along with strong constitutive promoter.
In another embodiment, the plasmid vector is carrying a DNA construct comprising a nucleotide sequence represented by SEQ ID NO.9.
In another embodiment, the present invention provides SEQ ID NO. 9. consisting of 5119 bp with Fe-Nitrogenase with strong promoter nifH (SEQ ID NO: 8) and integration site sequence formate dismutase with SEQ ID NO: 6 and 7.
In another embodiment, the plasmid vector is carrying a DNA construct comprising a nucleotide sequence represented by SEQ ID NO: 1.
In another embodiment, the plasmid vector is expressed in miro-organism selected from the group comprising of endophytic bacteria, epiphytic bacteria, rhizospheric bacteria and fungi.
In another embodiment, the bacteria is selected from the group comprising ofcultivable bacteris (viz) Methylobacterium extorquens; Beijerinckia indica; Azoarchus communis and uncultivable bacteria adapted to cultivabe using Ichip method (viz) Pseudomonas sp.; Bacillus sp. and Sphingomonas sp.
In another embodiment, the present invention provides a process for the preparation of genetically modified micro-organism(s) for improved nitrogen fixation and its delivery to crop-plants for assimilation, wherein the said microorganism is an endophyte, an epiphyte and a rhizospheric microbe, wherein the
said micro-organism is uncharacterized and non-cultivated, wherein the said modiciation is carried out by homologous recombination
In another embodiment, the present invention provides microbes selected from nitrogen fixing bacteria.
The Nitrogen fixing bacteria may include Proteobacteria (such as Methylobacter, Erwinia, Acinetobacter, Beijernickia, Sphingomona, Novosphingobium, Ochrobactrum, Gluconacetobacter etc.), Firmicutes (such as Clostridium sp. etc.), Cyanobacteria (such as cyanobacteria sp.), Actinobacteria (such as Frankia, Arthrobacter, Agromyces, Corynebacterium, Mycobacterium, Micromonospora, Propionibacteria etc.) and Bacteroidetes (such as Flavobacterium etc).
In another embodiment, the present invention provides the genetic modification of micro-organisms for improving Nitrogen fixation in plants.
In one more embodiment, the present invention provides genetically modifies microbes, wherein glucose uptake reduction is facilitated by ptsG gene (glucose transporter) disruption and amino acid catabolism is promoted by glnA gene (glutamine synthetase) disruption.
The microorganisms can be selected from but not limited to Methylobacterium, Methyloversatilis, Sphingomonas, Bosea, Altererythrobacter, Brevundimonas, Rubrivivax, Niveispirillum, Dinoroseobacter shibae.
Other microbes includes Nitrosopumilus maritimus , Nitrospira inopinata, Rhodobacter sphaeroides, Cupriavidus necator, Nigrospora oryzae, Azospirillum lipoferum, Rhodopseudomonas palustris , Bradyrhizobium japonicum , Ralstonia eutropha , Cyanobacteria, Epichloe typhina , Rhodococcus ,Xanthobacter species Nitrosomonas, Nitrosospira, Nitrosococcus, and Nitrosolobus .
In a further embodiment, the present invention provides certain fungi comprising Aspergillus, Candida, Chlamydomonas, Chrysosporium, Cryotococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora, Penicillium (e.g. P. chrysogenum), Pichia, Saccharomyces, Trichoderma and Xanthophyllomyces to be selected for genetic modification.
Further, Clostridium acetobutylicum, C. Beijerinckii, C. accharoperbutylacetonicum, C. saccharobutylicum, C. aurantibutyricum, C. tetanomorphum), Zymomonas, Escherichia (e.g., E. Coli), Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, Zymomonas and Saccharomyces, e.g., Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Kluyveromyces lactis, Saccharomyces lactiss.
In a further embodiment, the present invention provides endophytes selected from the group comprising Proteobacteria (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Se"atia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Delftia, Bradyrhizobiun, Sinorhizobium and Halomonas)' , Firmicutes (such as Bacillus, Paenibacillus, Lactobacil/us, Mycoplasma, and Acetabacterium) and Actinobacteria (such as Streptomyces, Rhodacoccus, Microbacterium, and Curtobacterium).
Bacteria that can be produced by the methods disclosed herein include Azotobacter sp., Bradyrhizobium sp., Klebsiella sp., and Sinorhizobium sp. The bacteria may be selected from the group consisting of Azotobacter vinelandii or Azotobacter chroococcum, Bradyrhizobium japonicum, Klebsiella pneumoniae, and Sinorhizobium meliloti. The bacteria may be of the genus Enterobacter and Rahnella. In another embodiment, certain fungi comprising Saccharomyces
cerevisiae and Trichoderma harzianum are selected for genetic modification for nitrogen fixation.
In another embodiment, the present invention provides a process for gene modification of microbes that reduces glucose transport and increases ammonia accumulation in crops by CRISPR/Cas technology.
In yet another embodiment, the present invention provides release of ammonia to the environment by the microbes in absence of glucose uptake.
Examples: Following examples are given by way of illustration therefore should not be construed to limit the scope of the invention.
Example 1: Down regulation/ Deletion of glnA, ptsG and amtB genes
Nitrogen fixation in diazotrophs was improved by reducing glucose uptake and increasing ammonia accumulation.
Genetic modifications were performed on ptsG gene, glnA gene (Sequence ID: 3) and amtB gene (Sequence ID: 4) in bacterial strains. The glucose uptake was reduced by disrupting the ptsG gene and the amino acid catabolism was promoted by disrupting the glnA gene (Sequence ID: 3) and the amtB gene (Sequence ID:4 )•
The gene modification comprised replacing the native promoter with a promoter with lower activity. This resulted in decreased expression or activity of these enzymes and thereby reduced glucose uptake and improved Nitrogen fixation and nitrate uptake by the plant.
The strain with downregulated Ammonia uptake was analysed for Ammonia release in comparison to unmodified wild type strain, under same conditions. Final strain with down regulated activity of Ammonia uptake, resulted in almost 2 fold increase in Ammonia release, which will have profound effect in field applications and very much beneficial to plants.
Example 2: Impact of downregulation of Ammonia uptake process on increased released of Ammonia: Inorder to increase the amount of Ammonia release from the cell, internal pathways which involve Ammonia uptake were manipulated. This includes Ammonia transporter for uptake of external ammonia and the Glutaminase/Glutamine synthase involved in fixing Ammonia in Amino acid Glutamine. Downregulation of genes was achieved by promoter exchange strategy, which resulted in 2 fold increase in Ammonia release by final strain when compared to wild type strain (figure 1).
Example 3: Enhancement of the Nitrogenase activity and regulator, for increased Nitrogen fixation in diazotrophs for enhanced Ammonia release to plants.
Gene modifications performed to increase Nitrogenase activity included, incorporation Fe-Nitrogenase (Sequece ID: 1) from microbes such as Azotobacter, Rhodobacter sp., Methylobacterium spp., for facilitating Molybdenum independent Nitrogenase activity.
To increase the nitrogenous activity, protein with highest nitrogenous activity was mined from available database using NCBI data server and bioinformatic approaches such as BLAST.
Steps of gene modifications of Gene
Inorder to overexpress Fe-Nitrogenase (Sequence ID: 1) copy of the heterologous gene was expressed under control of high strength nifH (Sequence ID: 8), constitutive promoter from Rhodobacter spp.,
Gene sequence coding for Fe-Nitrogenase (Sequence ID: 1) from Rhodobacter sp. was cloned by Gene synthesis method flanking sequence ID 6 and 7 as overlapping integration sites.
Expression cassestte was integrated into Methylobacterium spp., genome using homologous recombination at formaldehyde dismutase (fdm) inorder to delete the fdm sequence as well as simultaneous integration of Fe -nitrogenasegene cassette.
Expression cassette was cloned inside Nael restriction digestion sequences available in fdm between to get the final integration cassette (Sequence ID: 9).
Final integration cassette was taken in gene cloning vector pUC57 (Genscript plasmid), by gene synthesis
Gene transformation by homologous recombination:
Methylobacterium spp., electrocompetent cells were transformed with Integration cassette plasmid by standard methods
Integration was targeted to be at fdm sites in geneome of Methylobacterium spp. Transformants were screened for integrants, by colony PCR. Confirmed strains wer isolated and stored properly.
Engineered strains were subjected to growth and Ammpnia Production assay at controlled conditions. Wild type cells were also experimented in similar conditions, to test the increased Ammonia production by using Nessler reagent.
Manipulations also include overexpression/ multi copy expression of Nitrogenase controller protein such as NifA. All these manipulations were incorporated on strain with down regulated Gin activity, and finally resulted in almost 4 fold increase in N2 fixation, in terms of Ammonia production.
Example 4: Impact of enhanced activity of Nitrogenase and related factors, on increased Nitrogen fixation and Ammonia release: N2 fixation by Nitrogenase depends on additional regulation controls such as NifA regulator. Increasing the Nitrogenase activity, in particular, Fe-Nitrogenase activity and multicopy expression of NifA resulted in more than 4 folds increase in N2 fixation in terms of Ammonia release (in mM) (figure 2 ).
Example 5: Enablement of Autorophism by introduction of pathways related to utilization of Cl compound.
Introduction of pathways related to utilization of Cl compounds such as Methane, CO2, Formaldehyde, Methanol etc as souce of carbon and energy, facilitates the microbe to be Autotrophic and reduces dependency on sugar and organic acid substrates, either from external supply or from the plants.
Gene modification include incorporation/ overexpression of enzymes involved in methane assimilation, such as Methane monooxygenase, alcohol oxidase/
dehydrogenase and CO2 fixation via Formate dehydrogenase (FDH), Rubisco, and entry into regular metabolic pathways, finally resulting in ATP, NADH generation.
Enhanced ATP, NADH regeneration and increase carbon metabolism positively impacts on increased N2 fixation and hence increased Ammonia release (figure 3).
SEQUENCE LISTING
<110> Fertis India Pvt. Ltd
<120> A process for improving biological Nitrogen fixation by microbes beneficial to crops
<130> 001
<140> 202141042544
<141> 2021-09-20
<160> 9
<170> Patentin version 3.5
<210> 1
<211> 888
<212> DNA
<213> Fe-Nitrogenase
<400> 1 atgggcaaac tccgtcagat cgccttctac ggcaaaggtg gtatcggcaa gtcgaccacc 60 tcgcagaaca ccctcgccgc gctggtcgag atgggtcaga agatcctcat cgtcggctgc 120 gaccccaagg ctgacagcac ccgtctgatc ctgaacacca agctgcagga caccgtgctg 180
cacctggccg ccgaggccgg ttcggtcgaa gatctggaag tcgaagacgt cgtgaaaatc 240 ggctacaagg gcatcaaatg caccgaagcc ggcggtccgg agccgggggt tggctgcgcc 300 ggccgtggcg tcatcaccgc gatcaacttc cttgaagaaa acggcgccta tgacgatgtg 360 gactatgtgt cctatgacgt tctgggcgac gtggtctgcg gcggcttcgc catgccgatc 420 cgtgaaaaca aggcgcagga aatctacatc gtcatgtcgg gcgagatgat ggcgctttac 480 gccgccaaca acatcgccaa gggcatcctg aaatatgcga actcgggcgg cgtgcgtctg 540 ggcgggctga tctgcaacga acgcaagacc gaccgcgagc tggaactggc cgaagcgctg 600 gccgccaagc tgggctgcaa gatgatccac ttcgtgccgc gcaacaacgt cgtgcaacat 660 gccgaactgc gccgcgaaac cgtgatccaa tacgatccga cctgcagcca ggcgcaggaa 720 taccgcgaac tggcccgcaa gatccacgag aactcgggca agggcgtcat cccgaccccg 780 atcacgatgg aagagctgga agagatgctg atggatttcg gcatcatgca atcggaagaa 840 gatcgcgaaa agcagatcgc cgagatggaa gccgcgatga aggcctga 888
<210> 2
<211> 1605
<212> DNA
<213> NifA regulator
<400> 2
atggatggaa gcctcaaggg cgcgtcgagc acacactggc gtacgcagga gatgttcctc 60 gtccaggagg tcatgaacct gatcggcaag ggcctgcgcc tggatcaggt cgcgcgagag 120 atgcttcatc tcctgtccga gatcgtcgga ctgaaccggg gacggatcgt cctgaaagac 180 cccgacggca agggacaccg catgcctac gcctatgggc tgacccgcga ggaggtcgct 240 cggggccgct acgcgccggg cgagggcatc accggacgcg tcatccagga cggtcacctc 300 atcatcgtgc aggacatcga caaggacccg accttcctcg ggcgggcggt ggcgcgggcg 360 cagcttcccc gcggcgccgt gtcgttcctg gcactgccga tccgcgttga tcacaggacg 420 gtcggcgcca ttgcctgcca ccgcatccgc caccgggagc gggcgctctc ggacgatctc 480 accatcctgc gcatcatcgc cacgatggtc agccagttgc tgaccctgaa cgagcgggtg 540 gagcagaaga cgcgggcgct cgaggaacac aacgacatgc tggcgcgcga gctgcgcatc 600 aagcgcgccc gttacggcat catcggaacc tcgcccgcgc tgctgcgcgc cctcgcccaa 660 gtggagaagg tcgcgagcgc gaccgcgagc gtgctgctgc tcggcgagtc cggaaccggc 720 aaggaactct tcgcccgggc gctccacct gcgagtccgc gccgagaccg gccctcatc 780 aaggtcaatt gcgcggcgat ccccgacagc ctgtcgaat ccgagctgt cgggcacgag 840 cgcggcgcct tcaccggggc cgtcgatgcg cgggccggct ggttcgagca ggcgagcggc 900 ggcacgatct tcctcgacga gatcggcgag atgccggcgg tcctccagac caagctgctg 960
cgcacacttc aggaggggac ggtcgtacgc ctcggcggca agcgcgagat ccgggtcgat 1020 atgcgcctcg tcgccgcgac gaaccgggat ctcgccaccg aggtcgccca tggccgcttc 1080 cgagaggact tattctaccg cctcaacgtc gtgccgatca ccctgcctcc gctggcggag 1140 cggcgcagcg acatccccga tctggtgctg cactcctca cccaggcgaa ccagaacaac 1200 cagcgcaacg tcaacctgac ccagggcgcg gtcacgcatc tcgcccgcca gtcctggccg 1260 ggcaacatcc gccagctcgc caactcatc gagcggctcg tgctgctggc aagcgaaggt 1320 gtctggacg ccgaggacgt gcgtccgatg ctcgaaggtg ccgtcgtcac ggcgcccccg 1380 ctgctcgctc ccgcctcggc cccgttcttc tcgcccgaac gggaagcggc gtgcgcgcg 1440 atgggcaccg tgcgccccta tctgccggcc gactcgcacg gccacgaaca gttgcgcatg 1500 gccctggccc aggccggcgg caataaaacg cgggcggccc agcgcctcgg gctcaccgaa 1560 cgccaatttt cctatcgctg gcgcaagctc cagccggcac cctga 1605
<210> 3
<211> 1410
<212> DNA
<213> Glutamine synthase (Gin)
<400> 3 atgaatacgg ccgccgatgt gctgaaggcc atcaaggaca atgacgtcaa gtacgtcgac 60
ttccgcttca ccgacccgcg cggcaagtgg cagcacgtga ccttcgacgt ctccctgatc 120 gacgaggaga tgtccagga cggcaccatg ttcgacggct cgtcgatcgc tggttggaag 180 gcgatcaacg aatccgacat gctgctgatg cccgatccgg tcacggcctg catggatccg 240 ttcttctcgg cctccaccat gtcgatcgtc tgcgacgtgc tcgagccctc gactggcgag 300 ccctatgccc gcgatccgcg ctcgaccgcc aagctcgccg aggcgtatct gcgctccacc 360 ggcatcggcg acacgatct tgtcggcccc gaggccgagt tctcgtgt cgacgacgtg 420 aagtcggcg ccgaccccta ccataccggc tccagctcg actcgaccga gctgccgacc 480 aacggcttca ccgattacga gggcggcaac ctcggccacc gggtgcagac caagggcggc 540 tacttccccg tcccgccgca ggattcggct caggacatgc gcggcgagat gctggctgcg 600 atgcagtcca tgggcgtgaa ggtcgagaag caccaccacg aggtggcgtc ggcccagcac 660 gaactcggca tgaagttcga cacgctgacc ctgctcgccg accacatgca gatctataag 720 tactgcatcc acaacgttgc gcagagctac ggcaagtccg cgaccttcat gcccaagccc 780 gtctacggcg acaacggctc gggcatgcac gtgcaccagt cgatctggaa ggacggcaag 840 ccgctgtttg ccggcgacaa gtatgccgac ctcagccagg aatgcctgtg gtacatcggc 900 ggcatcatca agcacgccaa ggcgctgaac gcctcacca acccgtccac caactcctac 960
aagcgtctgg tgccaggcta cgaggccccc gtgctgctgg cctatcggc ccgcaaccgc 1020 tcggcctcct gccgtatccc gtggacgacg aacccgaagg ccaagcgcgt cgaggtccgc 1080 ttcccggatc cgatggccaa cccctacctc gcctctcgg cgctgctgat ggccggcctc 1140 gacggcatca tcaacaagat cgatccgggc ccggcgatgg acaaggatct ctacgacctg 1200 cccccgcgcg agctgaagaa gatcccgacc gtctgcggct cgctcgtga ggcgctgcag 1260 aacctcgaca aggaccgcgc ctcctcaag gccggcggcg tgtctcgga cgaccagatc 1320 gactcgttca tcgagctgaa gatggccgag gtgctgcgct acgagatgac cccgcacccg 1380 atcgagttcg tgcagtacta ctcgctgtaa 1410
<210> 4
<211> 1497
<212> DNA
<213> Ammonium transporter (Amt)
<400> 4 atgaaacttc gtaatcttct cgcgctcgga ctgggaggag ccgcccttgg cctcctcttc 60 gtcgagccgt cgctcgcgca ggcgccggcg gtcgagcccg ccgtcaccgc ggccgcaccg 120 gtgcccaaca agggcgacac ggcgtggatg ctgctctcgg cgatcctcgt cctgctgatg 180 accgtacccg gcctcgccct gttctacggc ggcctcgtgc gcaccaagaa catgctctcg 240
gtgctgaccc agatcttcgc gatcgcctcg atcgtctgcc tgctgtgggt gacctacggc 300 tacagcctcg ccttcaccaa tggcggcggc ctcaatgact tcgtcggcgg cttctcgaag 360 gcctcctca agggcgtcga cgccaactcg gtggcggcca ccttctccaa cggcgtcgtg 420 atccccgaat acgtctacat ctgcttccag atgacgttcg cgatgatcac ccccggcctc 480 atcgtcggcg cctcgccga gcggatgaag ttctcggcgc tggtcgtgt cacgatcctc 540 tgggtcacgc tgatctact cccgatggcc cacatggttt ggtactgggg cggtccggac 600 gtctcgccg acgcggcccg caagctcgcc gccgccggtg gcgaggccaa tgccgcggcc 660 aaggccgagt acgacgcggt gctgggcgat gccggcatgc tcttcaagtg gggcgccctc 720 gactcgccg gcggcaccgt cgtgcacatc aacgcaggca tcgccggcct cgtcggctgc 780 ctgatgctcg gcaagcgcat cggctacggc cgcgacctgc tggctccgca ctcgctgacc 840 atgaccacga tcggcgcctc gctgctctgg gtcggctggt tcggctcaa cgccggctcg 900 aacctcgaag ccaacggcgc cgcgggtctg gcgatgatca acacctcgt tgccaccgcc 960 gctgctgccg tctcctggct gtcgtggaa tgggccgcca agggtaagcc gtcgctcctc 1020 ggcatgctct cgggcgccat tgccggcctc gtcgccgtca ccccggccgc cggctttgcc 1080 ggcccgatgg gctcgatcgt tctgggtctg gccgccggcg cgatctgct cgtgatgtgc 1140 tccaccgtga agaacgcgct gggctacgac gactccctcg acgtgttcgg cgtgcactgc 1200
atcggcggca tcctcggtgc catcgccacg ggtatcctgg tctcgcccga tctcggcggc 1260 gccggcatcc ccgactacac caccaagccc ggtgagctga ccgtcggtgc ctacgacatg 1320 gccgcccagg tcatcatcca ggcgaaggcg gtgggcttca ccatcctgtg gtccggcatc 1380 ggctcggcga tcctctacaa gctcgtcgat ctgacgatcg gcctgcgcgt gacgcaggaa 1440 gaagagcgcg agggcctcga catcgccgac cacggcgagc gcgcctacaa ctactga 1497
<210> 5
<211> 2853
<212> DNA
<213> Formate dehydrogenase (fdh)
<400> 5 atgaccctca tcaaggaaat cgactacggc acgccgatcc ggctcaatga gcagacggtg 60 acgctgacca tcgacggcga gagcgtgacc gtgcccgcgg gcacctcggt catggccgcc 120 gcgatgcata tgggcaccaa gatcccgaag ctctgcgcta cggattcgct cgagccgttc 180 ggctcctgcc ggatgtgcct cgtggagatc gacggccgcc gcggcgcgcc cgcctcctgc 240 accaccccgg ccgagaatgg catggtcgtg cacacgcaaa ccgacaagct gcatcggctg 300 cgcaagggcg tgatggagct ctacatctcc gaccacccgc tcgactgcct gacctgcgcc 360 gccaacggcg attgcgagct gcaagatact gcaggtcagg tcggcctgcg cgaggtgcgc 420
tacggctatg acggcgacaa ccacgtcaag ccggcctccg accgctacct gcccaaggac 480 gagtcgaacc cctacttcac ctacgatccg tcgaagtgca tcgtctgcaa ccgctgcgtc 540 cgcgcctgcg aggagacgca gggcaccttc gcgctgacca tcgagggccg cggctcgac 600 agccgcgtgg cggcgggccc gaccaactc atgcagtccg aatgcgtgtc ctgcggcgcc 660 tgcgtgcagg cctgcccgac cgcgacgctg caggagaaga cgatccacca atacggccag 720 ccggaccatt ccgaggtcac gacctgcgcc tatgcggcg tcggctgcgc cttcaaggcc 780 gagatgcagg gcgacaaggt tgtccgcatg gtgccctaca agggcggcaa ggccaacgag 840 ggccatagct gcgtcaaggg ccgcttcgcc tacggctacg ccacccacaa ggaccgcatc 900 accaagccga tgatccgtga gaagatcacg gatccgtggc gcgaggtgtc gtgggaggag 960 gcgatcaacc acgccgcctc cgagttcaag cgcatccagg cgacgtatgg ccgcgactcg 1020 gtcggcggca tcacctcgtc gcgctgcacc aacgaggaag cctacctcgt ccagaagctg 1080 gtgcgcgccg ccttcggcaa caacaacgtc gatacctgcg cccgcgtctg ccactcgccg 1140 accggctacg gcctgatgtc cacgctcggc acctccgccg gcacgcagga ctcaagtcg 1200 gtcgaggaat ccgacgtgat cctcgtcatc ggcgccaacc cgaccgacgg tcaccccgtc 1260 ttcggctcgc ggatgaagaa gcggctgcgt gaaggcgccc gcctcatcgt cgccgacccg 1320
agaaaaatcg acctcgtgaa gtcgccccac atccgggccg accatcacct gccgctcaag 1380 cctggctcca acgtcgcctt catcaacgcc ttcgcccacg tcatcgtcac ggaagggctg 1440 atcgccgagg actacgtccg cgagcgctgc gatctggccg agttcgagtc ctgggcccgg 1500 ttcatcgccg aggagcgcaa ctcgccggag gccgcgcagg ccatcaccgg cgtcgatccg 1560 caggagatcc gcgccgcggc ccggctctac gccaccggcg gcaaggcggc gatctactac 1620 gggctcggcg tgaccgagca cagccagggc tcgaccatgg tgatgggcat ggccaacatc 1680 gccatggcca ccggcaatat cggcatggtg ggtgcgggcg tgaacccgct gcgcggccag 1740 aacaacgtgc agggctcctg cgacatgggc tcgttcccgc acgagctgcc gggctaccgc 1800 cacgtctcgg acgacgccac ccgcgagagc ttcgaggcga tctggggggc caagctcgac 1860 aacgcgccgg gcctgcgcat caccaacatg ctggacgagg ccgtcggcgg cagctcaag 1920 ggcatgtaca tccagggcga ggacatcgcg cagtccgacc ccgacaccca tcacgtcacc 1980 tccggcctca aggccatgga gtgcatcgtg atccaggacc tgtcctgaa cgagaccgcc 2040 aaatacgccc acgtctcct gccgggcgcc tcgttcctgg agaaggacgg caccttcacc 2100 aatgccgagc gccgcatctc ccgcgtgcgc aaagtgatgg ccccgatggg cggctacggc 2160 gattgggagg gcacggtgct gctcgccaac gcgctcggct acaagatgga gtacacccac 2220 ccgtccgaga tcatggacga gatcgcggcg ctcaccccga gctcgccgg cgtctcctac 2280
gacaagctgg aggagctggg ctcgatccag tggccgtgca acgagaaggc gccgctcggc 2340 acgccgatga tgcacgtcga ccggttcgtg cgcggcaagg gccgctcat gatcacggaa 2400 tacgtgccca ccgacgagcg gaccacgggc aagttcccgc tgatcctcac cacgggccgc 2460 atcctctcgc agtacaatgt cggcgcgcag acgcggcgga ccgagaactc ccgctggcac 2520 gaggaagacg tgctggagat ccacccctc gacgcggaga tgcgcggcat cgtcgatggc 2580 gacctcgtcg ccctggagag ccgctcgggc gacatcgcgc tgaaggccaa ggtgaccgag 2640 cggatgcagc cggggatcgt ctacacgacg tccaccacg ccaagaccgg cgccaacgtc 2700 atcaccaccg actattcgga ctgggcgacc aactgccccg aatacaaggt gacggcggtg 2760 caggtgcggc gcacgaaccg gccctccgac tggcaggcga agttctacga ggaggacttc 2820 tcgctcaccc gcatcgccga agcggcggaa tag 2853
<210> 6
<211> 300
<212> DNA
<213> Artificial
<220>
<223> Artificial sequence
<400> 6
atgcgtgcac tggtgtggca cggaacccag gacgtccggt gcgactcggt tcctgatccg 60 gagatcgagc acgagcgcga cgccatcatc aaggtcacga gtgcgccat ctgcggctcg 120 gacctgcacc tgtcgacca tttcataccc acgatgaagt cgggcgacat cctcggccac 180 gagaccatgg gcgaggtggt cgaggtgggc tcggcggcca agtccaagct caaggtcggc 240 gaccgggtgg tgatcccct cacgatcatc tgcggcgcat gcgaccagtg caagcgcggc 300
<210> 7
<211> 300
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence
<400> 7 gtcctgcgcg agatgatcta tgtctgccgg cccgccggca cgctctcggt gcccggcgtc 60 tatggcggcc tcatcgacaa gatcccgttc ggcgcgctga tgaacaaggg cctgacgatc 120 cgcacgggcc agacccacgt caatcgctgg agcgacgacc tgctgcggcg gategaggag 180 ggteagateg atccctcctt cgtgatcacc cataccgagc cgctggagcg cgggcccgag 240 atgtacaaga ccttccgcga caagcaggac ggetgeatea aggtegtget caagccctga 300
<210> 8
<211> 200
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence
<400> 8 ggcccgacaa aaccccgccc gacaccgcag gccctacaat ttgtcggcct tgttccattt 60 cgaacaaaac cttcaacacc atgatttcgc gtcattattt gcgaaaattc cggttggcac 120 gatggctgct gtagaagctg tgagcccggt taggaaccgt ctcgatattc gtgaagcacc 180 aacccccaag ggagccacac 200
<210> 9
<211> 5119
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence
<400> 9 atgcgtgcac tggtgtggca cggaacccag gacgtccggt gcgactcggt tcctgatccg 60 gagatcgagc acgagcgcga cgccatcatc aaggtcacga gttgcgccat ctgcggctcg 120
gacctgcacc tgtcgacca tttcataccc acgatgaagt cgggcgacat cctcggccac 180 gagaccatgg gcgaggtggt cgaggtgggc tcggcggcca agtccaagct caaggtcggc 240 gaccgggtgg tgatcccct cacgatcatc tgcggcgcat gcgaccagtg caagcgcggc 300 ggcccgacaa aaccccgccc gacaccgcag gccctacaat ttgtcggcct tgttccatt 360 cgaacaaaac cttcaacacc atgatttcgc gtcatattt gcgaaaatc cggttggcac 420 gatggctgct gtagaagctg tgagcccggt taggaaccgt ctcgatattc gtgaagcacc 480 aacccccaag ggagccacac atgacccgca agatcgccat tacggcaaa ggtggtatcg 540 gcaaatcgac gaccacccag aacaccgccg cagccctgc ctttttccac gaaaagaacg 600 tcttcatcca cggctgtgac ccgaaagccg acagcacccg gctgatcctg ggcggtctgc 660 cgcagcagac ggtgatggac acgctgcgca tcgagggcgc cgagcgcgtc accgtggaca 720 aggtcgtgaa gaccggcttc aaggacatcc gctgcgtgga atcgggcggg ccggagccgg 780 gcgtgggctg cgccggtcgc ggcgtcatca ccgccatcga cctgatggaa gaaaacgaag 840 cctacagcga agaccttgat tcctgttct tcgacgtgct gggcgacgtg gtttgcggcg 900 gcttcgccat gcccatcgc gacggcaagg cggaggaagt ctatatcgtc gcctcgggcg 960 agatgatggc gatctatgcc gccaacaaca tctgcaaggg tctggcgaaa tacgcccggc 1020 aatcgggcgt gcgtctgggc gggatcatct gcaacagccg caatgtcgat ggcgaaaagg 1080
aatcctga ggaatcacc aaggccatcg gcaccaagat gatccactc gtgccgcgcg 1140 acaacatcgt gcaaaaggcc gagttcaaca agcagaccgt gaccgaatc cagcccgagg 1200 ccaatcaggc gcaggaatac cgcgaactcg gccgcaagat catcgagaac gaggatttcg 1260 tgatcccgaa gccgctcgcc atggatgagc tggaagccat ggtcgtcaaa tacggcctga 1320 tggactgatc ctccggggcg ccgcgcccgg cgccctttcc ccgaatttcc atggagttag 1380 gccgatgccc taccatgagt ttgaagtctc gaaatgcatc cccgagcggc gcgagcacgc 1440 ggtgatgaag gcggcgggcg aggatctgac ctctgcctg ccgaaaggct acctgaacac 1500 gatccccggc acgattccg aacgcggctg cgcctattgc ggcgcaaaac acgtgatcgg 1560 cacgccgatg aaggacgtga tccacatctc ccacgggccc aatggctgca cctatgacac 1620 ctggcagacg aagcgctaca tctcggacaa tgacaacttc cagttgaaat acacctttgc 1680 gaccgacgtg aaggaaaagc acgtcgtctt cggcgccgag gggctgctga agaaatcgat 1740 gcacgaggcc tttgacgcct tcccgaacat caagcggatg acggtctatc agacctgcac 1800 gacggcgctg atcggcgatg acgtcgatgc catcgccaag gaagtgatgg aagaacgcgg 1860 cgatgtcgat gtgttcgtct gcaactcgcc cgggttcgcg gggcccagcc aatcgggcgg 1920 tcaccacaag atcaacatcg cctggctgaa ccagaaagtc ggcacggtcg agccggacta 1980
cctaggcgaa cacgtcatca actacgtggg cgaatacaac atccagggcg accaggaggt 2040 gatgatcgac tatttcaacc gcatgggcat tcaggtgctg tcgaccttca ccggcaacgg 2100 cagctatgac agcctgcgga tgatgcaccg cgcgcatctg aacgtgctcg aatgcgcgcg 2160 gtctgccgaa tacatctgcg acgaactccg cgcccgctac ggcattcccc ggctggatat 2220 cgacggcttc ggctcgagc cgctcgccaa ttcgctgcgc aaggtcgcgc tctcttcgg 2280 catcgaggac aaggccgagg cgatcatcgc cgaggaatat gcgaagtgga agccgcagct 2340 ggactggtac aaggaacggc tgaagggcaa gaaggtctgc ctctggccgg gcggatcgaa 2400 gctctggcac tgggcccatg cgatcgaaga ggaaatgggc ctcaaggtcg tgtcggtcta 2460 taccaagttc ggccatcagg gcgacatgga aaagggcgtc tcgcgttgcg gcgagggggc 2520 ctggcgatc gacgacccga acgagctgga atcggtcgaa gccatcgaga tgctgaagcc 2580 cgacatcatc tcaccggca aacgccccgg cgaatcgtc aagaaacacg gcgtccccta 2640 tctgaacgcc catgcctatc acaacgggcc tacaagggc tcgagggct gggtccgct 2700 cgctcgcgac atctacaacg cgatctatc gccgatgcgg cagctggcgg cgctggatat 2760 ttccgccccc gatgcggcca tcacctcggg cttccgcacc gccaagatga acgccgatct 2820 gaccgtttcg gatgaggtca agttcagcga ggtgctgcac gaatacaccg gcaaatacga 2880 ctcgattgcc gagatccgcg cccgcaatca ggcctatgcc gccgagcaga aagcgctccg 2940
cgacgccgt caacctgccg ccgaatgagg gacagatgac cgatatcagc gaaaaactcg 3000 atccgctcgt cgattacatc atgaagaact gcctgtggca gtcaactcg cgcggctggg 3060 accggctcaa gcagaatgcc gggatcctgt cgcagacctg cgagatcctg tgcggcgagg 3120 agccggtgca tgaaaccgcg atggaccgct gctactgggt cgatgcggtg atcctgtcgc 3180 gcgcctacaa ggcccgcttc ccctggctga tggccatgac caagcccgag atcaagagcc 3240 tgttcaaggc gttgcacgag aagatcgacc atctcaccgt tcacggctcg ctcaataccg 3300 agctgaccgt cccgcattat tgaaaggaag accccaatga ctgccaggt cacccagaag 3360 gcgcgggaag gcacgatcaa cccgatcttc acctgccagc ccgccggggc gcaattcgcc 3420 tcgatcggga tcaaggatg catcggcatc gttcacggcg gccagggctg cgtgatgtc 3480 gtgcgtctgc tgatctcgca gcacatgaag gaaagctcg agatcgcctc gtcgtcggtc 3540 catgaagatg gcgcggtctt cggcgcgctc gaccgtgtcg aaaccgcggt cgaggtgct 3600 ttgacccgct accccgatgt gaaggtggtg ccgatcatca ccacctgctc gaccgagatc 3660 atcggcgacg acgtggacgg gctgctttcg aaactcgaag acgagttgct gccgaccaaa 3720 ttccccggcc gcgaagtgca tctgctcacc gtgcatgcc cgagcttcgt tggctcgatg 3780 atcaccggct atgacaaggc ggtgcatgat ttcgtgaaga aatcgcgac gaaggacgag 3840
cccagcgaca agatcaacct gatcaccggc tgggtgaacc cgggcgatgt gaaggagctg 3900 aaacaccttc tggaggtgat ggaggtcaag gcgaacgtgc tctttgaagt cgaaagcttc 3960 gacagcccgc tgatgcccga tctggaacac cattcgcacg gctcgaccac gatcgaggat 4020 ctgcgcgaca cggccaatgc gaaaggcacc atcgcgctca atcgctatga aggcatgaag 4080 gccgccgat acctgaagaa gaagttcaag gttcccgcgg tgatcgggcc gaccccggtc 4140 ggcatccgca acaccgatgc cttcctgaaa gcggtctccg agatgaccgg ccagccgatc 4200 ccggcgcagc tggtcaagga acggggcct gcgctcgatg ccatcgccga catcggccac 4260 atgtttctgg ccgacaaaag ggtggcgatc tatgcgaacc ccgatctggc catcggcctg 4320 accgagttct gccttgacct cgaaatgaag ccgaaactgc tgcttctggg cgatgacaac 4380 tcgggctatg tgaaagaccc ccgcgtgctg gcgctgcagg aaaacgcgcc ggatctggaa 4440 atcgtgacga acgccgattt ctgggatctg gaaagccgca tccagcaagg gctcgaactc 4500 gatctgatcc tcggccattc caagggccgg tcatctcga tcgactacaa ggtgccgatg 4560 gtccgcgtgg gctcccgac ctacgaccgg gcagggatgt atcgccatcc ggtgctgggc 4620 tacggcgggg cgatgttcct tgccgaaacc atggccaaca cgcttttcgc cgacatggag 4680 gcgaagaaaa acaaggaatg gatcctcaac gtgtggtgac gtctgaagcc ggaactgcct 4740 gacggcagat gcgaaccgtc cctcccccgc tcacccgggg gagggacggg gccgagatgg 4800
cgcgggcacg aacggcctgg tcctgcgcga gatgatctat gtctgccggc ccgccggcac 4860 gctctcggtg cccggcgtct atggcggcct catcgacaag atcccgttcg gcgcgctgat 4920 gaacaagggc ctgacgatcc gcacgggcca gacccacgtc aatcgctgga gcgacgacct 4980 gctgcggcgg atcgaggagg gtcagatcga tccctccttc gtgatcaccc ataccgagcc 5040 gctggagcgc gggcccgaga tgtacaagac cttccgcgac aagcaggacg gctgcatcaa 5100 ggtcgtgctc aagccctga 5119
Claims
1. A composition comprising genetically modified micro-organism(s) consisting of upregulation of nifA and iron containing Nitrogenase and downregulation of glutaminase gene for increased Nitrogen fixation wherein the said micro-organism is an endophyte, an epiphyte and a rhizospheric microbe.
2. The composition as claimed in Claim 1, wherein the composition comprising genetically modified micro-organism(s) consisting of down regulation or deletion of gln and amtB gene.
3. The composition as claimed in Claim 1, wherein the composition comprising genetically modified micro-organism(s) consisting of disruption of ptsG gene, glutamine synthase gene, glnA gene and Ammonia transporter gene, amtB gene.
4. The composition as claimed in claim 1, wherein the plasmid vector or suicidal plasmid consists of integration site as well as over expression genes along with strong constitutive promoter.
5. The composition as claimed in claim 2, wherein the plasmid vector of is carrying a DNA construct comprising a nucleotide sequence represented by SEQ ID NO.9
6. The composition as claimed in claim 4, wherein SEQ ID NO. 9. consisting of 5119 bp with Fe-Nitrogenase with strong promoter nifH (SEQ ID NO: 8) and integration site sequence formate dismutase with SEQ ID NO: 6 and 7.
7. The composition as claimed in claim 2, wherein the plasmid vector is carrying a DNA construct comprising a nucleotide sequence represented by SEQ ID NO: 1
The composition as claimed in claim 1, wherein the said plasmid vector is expressed in miro-organism selected from the group comprising of endophytic bacteria, epiphytic bacteria, rhizospheric bacteria and fungi. The composition as claimed in claim 1, wherein the bacteria is selected from the group comprising of cultivable bacteris (viz) Methylobacterium extorquens; Beijerinckia indica; Azoarchus communis and uncultivable bacteria adapted to cultivabe using Ichip method (viz) Pseudomonas sp.; Bacillus sp. and Sphingomonas sp. A process for the preparation of genetically modified micro-organism(s) for improved nitrogen fixation and its delivery to crop-plants for assimilation, wherein the said micro-organism is an endophyte, an epiphyte and a rhizospheric microbe, wherein the said micro-organism is uncharacterized and non-cultivated, wherein the said modiciation is carried out by homologous recombination
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110012170A (en) * | 2009-07-30 | 2011-02-09 | 전북대학교산학협력단 | Host cells transformed with hydrogenase or nitrogenase of rhodobacter sphaeroide and method for preparing hydrogen using the same |
| WO2017011602A1 (en) * | 2015-07-13 | 2017-01-19 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| KR20180082404A (en) * | 2018-07-10 | 2018-07-18 | 경북대학교 산학협력단 | Novle Rhodobacter sphaeroides KNU-04 strain having plant growth promoting effect, and uses thereof |
| WO2020014498A1 (en) * | 2018-07-11 | 2020-01-16 | Pivot Bio, Inc. | Temporally and spatially targeted dynamic nitrogen delivery by remodeled microbes |
| WO2020219932A1 (en) * | 2019-04-24 | 2020-10-29 | Pivot Bio, Inc. | Gene targets for nitrogen fixation targeting for improving plant traits |
| WO2020245841A1 (en) * | 2019-06-04 | 2020-12-10 | Fertis India Pvt. Ltd. | Genetic modification of endophytic/epiphytic/rhizospheric microbes for improved nitrogen fixation for crops |
-
2023
- 2023-03-20 WO PCT/IN2023/050271 patent/WO2023181067A1/en unknown
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110012170A (en) * | 2009-07-30 | 2011-02-09 | 전북대학교산학협력단 | Host cells transformed with hydrogenase or nitrogenase of rhodobacter sphaeroide and method for preparing hydrogen using the same |
| WO2017011602A1 (en) * | 2015-07-13 | 2017-01-19 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| KR20180082404A (en) * | 2018-07-10 | 2018-07-18 | 경북대학교 산학협력단 | Novle Rhodobacter sphaeroides KNU-04 strain having plant growth promoting effect, and uses thereof |
| WO2020014498A1 (en) * | 2018-07-11 | 2020-01-16 | Pivot Bio, Inc. | Temporally and spatially targeted dynamic nitrogen delivery by remodeled microbes |
| WO2020219932A1 (en) * | 2019-04-24 | 2020-10-29 | Pivot Bio, Inc. | Gene targets for nitrogen fixation targeting for improving plant traits |
| WO2020245841A1 (en) * | 2019-06-04 | 2020-12-10 | Fertis India Pvt. Ltd. | Genetic modification of endophytic/epiphytic/rhizospheric microbes for improved nitrogen fixation for crops |
Non-Patent Citations (1)
| Title |
|---|
| WILLISON ET AL.: "Sequence and transcript analysis of the nitrogenase structural gene operon (nifHDK) of Rhodobacter capsulatus: evidence for intramolecular processing of nifHDK mRNA", GENE, vol. 133, no. 1, 29 October 1993 (1993-10-29), pages 39 - 46, XP023540187, DOI: 10.1016/0378-1119(93)90222-O * |
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